1. Field of the Invention
The present invention relates generally to methods for the electrophoretic separation and analysis of oligosaccharides. More particularly, the present invention relates to analytical methods involving electrophoretic based profiling of oligosaccharides following their chemical or enzymatic release from glycoproteins.
2. Description of Relevant Art
Complex carbohydrates are important components of all living things. In addition to providing energy and structural supports for cells, increasing evidence has shown that the carbohydrate moieties of glycoconjugates are often important as recognition determinants in receptor-ligand or cell-cell interactions, in the modulation of immunogenicity and protein folding, and in the regulation of protein bioactivity. Changes in the biological activity of glycoproteins often result from alterations in protein glycosylation either through variable site occupancy or changes in the structure of the oligosaccharide occupying a particular site.
For these reasons biochemists have increasingly recognized the benefits associated with the ability to obtain molar ratios or patterns of oligosaccharide structures of glycoproteins. It is equally desirable to have the ability to determine the degree of glycosylation and to detect changes in the extent of and the nature of the glycosylation of proteins.
Recently there have been rapid developments in methodologies for analyzing oligosaccharide structures of native and recombinant glycoproteins. Anion exchange chromatography, reverse phase high pressure liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy and more recently electrophoretic methods have all been investigated for their usefulness in profiling the oligosaccharide structures of glycoproteins with varying degrees of success. The most common methods for profiling asparagine linked (N-linked) oligosaccharides involve releasing the oligosaccharides with peptide-N-glycosidase F(PNGase F) and profiling the released oligosaccharides by high pH anion exchange chromatography or slab gel electrophoretic separations.
Profiling information obtained from high pH anion exchange chromatography depends upon the ionic strength of the released oligosaccharides, and, because some molecular sieving takes place, it also depends upon the effective or apparent molecular weight of the oligosaccharides. While effective molecular weight is a measure of molecular size, effective molecular weight primarily depends upon the oligosaccharide hydrodynamic volume. Hydrodynamic volume is determined by the oligosaccharide molecular weight and the degree to which the oligosaccharide is hydrated by the medium in which the oligosaccharide is being analyzed. Thus, different oligosaccharides which have been analyzed by high pH anion exchange chromatography may have the same molecular weight or molecular size but will appear to have different molecular size because the degree to which they are hydrated is different. Reliable molecular size correlation data which takes into account the relative amount of hydration experienced by different saccharides, is available. However, this data depends upon a qualitative knowledge of the oligosaccharide being investigated. Also, oligosaccharides released from glycoproteins frequently contain one or more sialic acid residues which can alter dramatically the charge and the hydrodynamic volume of the oligosaccharide. In these cases, high pH anion exchange chromatography provides little reliable molecular size information. Thus, to obtain reliable relative oligosaccharide molecular weight information from high pH anion exchange chromatography data it is preferable to know the degree to which the glycoprotein is sialylated.
Electrophoretic based separations for profiling oligosaccharides represent an improvement over high pH anion exchange chromatography because electrokinetic migration of any given compound is primarily dependent upon only its mass to charge ratio. Typically, these electrophoretic methods utilize high pH (greater than 8.3) borate buffer electrophoretic mediums. The borate complexes with the oligosaccharide hydroxyl moieties providing the charge required for electrokinetic movement. One disadvantage associated with these borate buffer systems is the electrophoretic migration pattern of the borate complexed oligosaccharides. These complexes will migrate in a reverse direction (i.e. large structure migrate first) rendering the identification of the structures imprecise and difficult. Typically, in order to achieve low detection limits with very small sample volumes characteristic of capillary electrophoretic separations, saccharides are pre-labeled with a fluorescent labeling compound at the saccharide reducing end. When the label is charged, the oligosaccharides have the same charge and will electrokinetically migrate according to their mass to charge ratio When the oligosaccharides are not sialylated, all of the structures possess the same charge and the resulting data provides relative size information about the separated and detected saccharides.
This approach presents problems when analyzing oligosaccharide structures of sialylated glycoproteins. Sialic acid becomes negatively charged at pHs of about 7 to 8. Under electrophoresis conditions which utilize running buffers having a pH above 8, the sialic acid component of the released oligosaccharide is ionized. In this case, in addition to the problems discussed above, each oligosaccharide may have different charge distributions, so the electrokinetic migration order of the sialylated compounds effects the mass to charge ratio. To obtain relative size information for such oligosaccharides, the cleaved compounds must be desialylated and then electrophoresed. In this manner the relative sizes of the desialylated oligosaccharides are known but information relating to the relative size of sialylated residues is not determined directly.
In cases involving glycoprotein conjugate profiling under conditions in which the electrophoresis conditions include a low pH buffer of about 2.5, the sialylated oligosaccharides tend to co-migrate resulting in very poor separations.
It is an object of the present invention to provide methods for profiling glycoprotein linked high mannose, complex and hybrid oligosaccharides. It is also an object of the present invention to provide methods for obtaining molar ratios and relative molecular weights of individual oligosaccharides. It is further an object of the present invention to provide methods for detecting changes in the extent or nature of glycosylation, including changes in the extent of sialylation.